Abstract Genes that regulate chromatin organization and function have been implicated in the development of skeletal growth disorders. A major gap in our current knowledge is how epigenetic regulators control longitudinal bone growth at the growth plate. From a child's health perspective, a deeper understanding of the role of specific chromatin modifiers in the postnatal growth plate cartilage may lead to more informed use of targeted epigenetic therapies that are under investigation for the treatment of pediatric cancers. The goal of our proposal is to establish the requirement of a chromatin modifier Dot1L (Disruptor of telomere silencing-like 1) in growth plate cartilage and to uncover transcriptional mechanisms through which it regulates the progression of chondrocyte differentiation. Dot1L is a unique chromatin modifier because it is the only known enzyme that catalyzes the methylation of lysine residue 79 in histone 3 (H3K79), an established epigenetic mark for gene transcription. A human genetic variant of Dot1L is associated with taller pubertal stature and accelerated growth rate in adolescent idiopathic scoliosis. Recently, inactivation of the Dot1L gene in embryonic cartilage was shown to cause runting, growth plate disorganization, and early death in mice (1, 2). While inhibition of Dot1L is considered a promising therapy for treatment of leukemia in children(3-6), we know little about the influence of Dot1L on postnatal growth plate function. Thus, we developed an inducible strategy to genetically disrupt Dot1L in postnatal growth plate chondrocytes in mice. Postnatal inactivation of Dot1L in growth plate cartilage using the tamoxifen-inducible Aggrecan-Cre driver (AgcCreERT2) resulted in an intriguing skeletal phenotype characterized by impaired longitudinal bone growth, structural changes within growth plate cartilage, and focal closure of the growth plate. Our preliminary in vitro studies further suggest that Dot1L modulates signaling through the bone morphogenetic protein (Bmp) pathway, a key pathway controlling chondrocyte differentiation and endochondral ossification. Together, our data reveal a critical and understudied role for Dot1L in maintaining postnatal GP cartilage. We hypothesize that the chromatin modifier Dot1L maintains the proper balance of chondrocyte proliferation and maturation to support postnatal bone elongation at the growth plate. In this study, we will use cutting-edge genetic and molecular approaches to advance our understanding of Dot1L-mediated regulation of growth plate chondrocyte differentiation and postnatal bone growth by the following Specific Aims: (1) determine whether postnatal function of Dot1L in the GP restricts chondrocyte maturation; and (2) identify Dot1L-regulated genes and networks controlling chondrocyte differentiation in the postnatal growth plate. This contribution is significant since it will advance our understanding of the epigenetic regulation of cartilage genes linked to human skeletal dysplasia and growth disorders. The workflow and data generated by funding this R03 proposal will lay the groundwork for future projects focused on establishing the epigenetic circuits necessary for skeletal growth, development and repair. !